Infections caused by gram-negative bacteria are an increasing health problem, particularly in hospitalized patients. Plasmids of these organisms determine a variety of beta-lactamases, enzymes that are responsible for resistance to penicillins, cephalosporins, and related beta-lactam antibiotics. Based on substrate profile, molecular weight, isoelectric point, and other properties, more than 20 plasmid-mediated beta-lactamases are presently known. As the major biochemical mechanisms for resistance to beta-lactam antibiotics it is important to understand how these enzymes are related genetically and how they are evolving in response to the changing selection pressure imposed by the introduction of new beta-lactam drugs. The aims of this proposal are: 1) To establish homology relationships among the various beta-lactamase genes by preparing specific probes from sequenced TEM-1 and CEP-1 genes and from OXA-1 and PSE-1 genes that we have cloned for hybridization against isolated restriction fragments determining the 21 beta-lactamase gene types. 2) To investigate the possible origin of beta-lactamase from endopeptidases involved in cell wall biosynthesis by hybridization studies between genes determining beta-lactamases and cloned genes determining penicillin binding proteins of Escherichia coli. 3) To compare the nucleotide sequence of selected beta-lactamase genes cloned in phage M13 vectors to determine the molecular details of their evolutionary relationships. 4) To elucidate structural features responsible for the activity of beta-lactamases against various beta-lactam substrates by comparing the sequences of enzymes with different substrate profiles, by sequence alterations through site-directed mutagenesis, and by the generation in vitro of beta-lactamase gene hybrids. An understanding of how beta-lactamase genes have evolved and have developed the capacity to hydrolyze new substrates should assist in the design of even more efficient beta-lactam antibiotics.